Endothelial progenitor cells derived from CD34+ cells form cooperative vascular networks.

Studies were conducted to investigate whether endothelial progenitor cells (EPCs) derived from CD34(+) cells could differentiate to endothelial- and smooth muscle-like cells, respectively. Differentiation was induced by either basic fibroblast growth factor (bFGF) or platelet-derived growth factor-BB (PDGF-BB). Further, the contribution of these cells to cooperative blood vessel formation was examined. Immunofluorescence microscopy and Western blot analysis were used to assess vascular smooth muscle (SMC) or endothelial (EC) cell differentiation. SMC (α-SM actin and calponin) or EC (CD31 and von Willebrand factor, vWF) markers showed significant expression. In contrast, expression of the stem/progenitor cell marker CD133 gradually decreased. To quantify the percentage of positive cells of smooth muscle cell marker and endothelial cell marker, flow cytometric analysis was performed, and the results demonstrated that up to 83.76% of PDGF-BB treated-cells were positive for α-SMA marker and up to 89.27% of bFGF-treated cells were positive for vWF marker. To assess functional properties of the endothelial- and smooth muscle-like cells and to demonstrate an association of the two cell types in vascular structures, we evaluated the capacity of these cells to form angiogenesis-like networks on Matrigel. Both endothelial-like cells plated in isolation and co-cultured with smooth muscle-like cells showed formation of vascular networks on Matrigel while smooth muscle-like cells alone did not. Co-culture of fluorescently labeled endothelial- (PKH26, red fluorochrome) and smooth muscle-like cells (PKH67, green fluorochrome) revealed that these cells formed cooperative networks of tubular structures on Matrigel. Collectively, our findings suggest that EPCs can differentiate into both smooth muscle- and endothelial-like cells and contribute to the development of cooperative vascular networks. Notably, this differentiation could also provide a stable foundation for maintenance of the newly formed vascular networks.